Complementary use of MALDI-TOF MS and real-time PCR-melt curve analysis for rapid identification of methicillin-resistant staphylococci and VRE.

Wai-Sing Chan, Tsz-Ming Chan, Tsz-Wan Lai, Jasper Fuk-Woo Chan, Raymond Wai-Man Lai, Christopher Koon-Chi Lai, Bone Siu-Fai Tang

Index: J. Antimicrob. Chemother. 70(2) , 441-7, (2015)

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Abstract

To develop a rapid method for routine screening of methicillin-resistant staphylococci and VRE for clinical isolates and positive blood cultures.Our method consisted of two parts: MALDI-TOF MS was used for identification of staphylococci and enterococci, followed by antibiotic resistance detection by real-time PCR-melt curve analysis without DNA extraction. The latter part included a triplex reaction for staphylococcal culture isolates (mecA, mecALGA251 and Panton-Valentine leucocidin genes), dual PCR of mecA/mecALGA251 and nuc genes for staphylococcal blood cultures, and a duplex reaction for enterococci (vanA and vanB genes). A total of 124 clinical isolates and 56 positive blood cultures were tested. MALDI-TOF MS was performed using Microflex LT (Bruker Daltonik, Bremen, Germany) and Rotor-Gene Q (Qiagen, Hilden, Germany) was used for real-time PCR-melt curve analysis. The total assay time was <2.5 h.The results revealed 100% concordance with antibiotic susceptibility testing or other reference methods for all culture isolates and enterococcal blood cultures. The percentage of concordance for staphylococcal blood cultures was 97.5%.The method described herein was fast, economical, reliable and capable of detecting mecALGA251, vanB1 and vanB2 genotypes, which are not included in most commercial assays. Large-scale screening is required to further test the performance of this protocol, especially for genotypes that are infrequently encountered.© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.